HSD enzymes belong to the group of NADP(H)/NAD(H) dependent oxidoreductases that regulate in the body the conversion of ketosteroids to hydroxysteroids and vice versa in a process called steroidogenesis. 17βHSD allow this oxidation-reduction process at the C-17 position of the steroid skeleton. Reduction of the C-17-oxo group of steroids turns a biologically not very active estrone (E1) into a highly potent 17β-estradiol (E2) as well as 4-androsten-3,17-dione into testosterone (T) and 5α-androstan-3,17-dione into dihydrotestosterone. These 17β-hydroxy forms, unlike 17β-ketosteroids, have a high affinity for the respective receptors and thus have a major influence on numerous processes in the body, especially the proliferation and differentiation of cells. 17βHSD therefore play a key role in the formation of biologically active estrogens and androgens and specific regulation of these enzymes could open the door to new groups of therapeutic and diagnostic agents (Poirier, D. Current. Med. Chem. 2003, 10, 453).
At present, 15 isoenzymes of the 17β-HSD family are already described in the literature, with individual types differing significantly from each other by tissue, substrate and cofactor specificity, as well as the distribution in cells.
Overexpression of some 17βHSD isoenzymes, and thus the excessive production of 17β-hydroxysteroids in tissues, correlates with the occurrence of estrogen/androgen-dependent diseases. Selective inhibitors of the individual isoenzymes could advantageously be used to treat such diseases, and the determination of the rate of expression of the respective types of HSD in the affected tissues could then serve as a diagnostic marker of said diseases. This is the reason for the considerable attention that has been devoted to 17βHSD research in recent decades.
The present invention relates to inhibitors of 17βHSD1 and 17βHSD5 isoenzymes. These isozymes are involved in the production of estrogens and androgens in the human body. They affect the progression of numerous hormone-sensitive diseases, particularly breast cancer, prostate cancer, non-small cell lung cancer (NSCLC), squamous cell carcinoma, and others. Elevated E2 levels result in benign anomalies, especially endometriosis, adenomyosis, uterine myomas, and menstrual cycle disorders. A great deal of effort is still dedicated to searching for substances that are capable of influencing the course of such illnesses. This is due either to the absence of an appropriate therapeutic substance or to the disadvantages of existing therapeutic options used in the treatment of hormone-sensitive diseases. These include, in particular, adverse effects, the emergence of resistance in long-term administration, and, last but not least, the frequent relapse of the disease.
17βHSD1 was the first of all isoenzymes to be described by Engel et al. in the 1950s (Ryan, K. J.; Engel, L. L. Endocrinology 1953, 52, 287). 17βHSD1 preferentially regulates the reduction of estrone (E1) to estradiol (E2) and is naturally expressed especially in placenta, ovaries, breast tissue, uterus and endometrium. It catalyzes the production of E2 from E1 in the ovaries; and in peripheral tissues, it controls the level and availability of E2 at the pre-receptor level (intrakrine modulation). To a lesser extent, 17βHSD1 also catalyzes the conversion of dehydroepiandrosterone (DHEA) to 5-androsten-3β,17β-diol (Δ5-diol).
The importance of the E2 sex hormone in the human body is its strong affinity to estrogen receptors (ER), through which it regulates the expression of a number of genes (so-called transcription factor). ER in idle state are usually found in the cytosol in the form of monomers. After binding E2 to ER, the receptor units dimerize, enter the cell nucleus and bind to the DNA sequence called the estrogen response element (ERE). After the E2-2ER complex is bound to ERE, a cascade of processes is activated leading to cell proliferation and differentiation. This multiplication of cells can be both physiological and pathological, leading to malignant proliferation (Ciocca, D. R.; Fanelli, M. A. Trends Endocrinol Metab. 1997, 8, 313).
It follows from the above that proliferation and differentiation of cells caused by estrogens can be regulated by influencing the metabolism of E2. One possibility is to avoid the binding of E2 to the active site of ER, another possibility is blocking the synthesis of E2 itself by inhibiting one or more enzymes involved in steroidogenesis (17βHSD1, aromatase, sulfatase) (Hong, Y.; Chen, S. Mol. Cell. Endocrinol. 2011, 340, 120).
Estrogen sensitive types of breast cancer, female genital tract cancer and lung cancer (non-small cell lung cancer—NSCLC) are characterized by an increased expression of 17βHSD1. Increased E2 levels are then associated with a number of benign anomalies. The most common of these include endometriosis (pathological localization of the uterine lining elsewhere than in the uterine cavity), adenomyosis (moving the uterine cavity lining—the endometrium—into the uterine muscle layer), menorrhagia (abnormally strong menstrual bleeding), metrorrhagia (acyclical, dysfunctional bleeding), dysmenorrhea (pain and other difficulties associated with menstruation) and uterine myomas (WO 2008034796 A2).
17βHSD5 is expressed mainly in the testes, it is commonly found also in the prostate, liver and adrenal glands. Some types of breast and prostate tumors have increased expression (Dufort et al. Endocrinology. 1999, 140, 568). Among other isoenzymes, 17βHSD5 has a somewhat special position. As the only one in the 17βHSD family of enzymes, it belongs to so-called aldo-ketoreductases, while the other types are dehydrogenases/short chain reductases. In addition, thanks to a very spacious binding site, it exhibits a certain substrate multispecificity. That is, although it preferentially reduces 4-androsten-3,17-dione to T, it also binds some other estrogens and androgens and affects their conversion at the 3α-, 17β- and 20α-positions. 17βHSD5 is also involved in the synthesis of prostaglandins (prostaglandin PGF2α). PGF2α has been shown to play an important role in the growth of some types of tumors, particularly colorectal carcinoma (Qualtrough, D. Int. J. Cancer 2007, 121, 734). Since the effect of 17βHSD5 has been demonstrated on the progression of both steroid-sensitive and non-sensitive carcinomas, selective inhibition of 17βHSD5 has long been one of the challenges for further research.
The most common cancer in women is breast cancer. The standard procedure for the treatment of early stages of estrogen-positive breast cancer types is surgery and subsequent adjuvant chemotherapy. In the context of subsequent therapy, selective estrogen receptor modulators (SERMs) such as Tamoxifen, Raloxifen and others are most often used in premenopausal women. These are partial or complete ER antagonists. In postmenopausal women whose tumors have ER+ status, Tamoxifen remains the drug of choice. In the case of ER− breast tumors, SEEMs (Selective Estrogen Enzyme Modulators) are a good choice, such as Tibolon or Anastrozole. These substances selectively affect the respective enzymes of steroidogenesis, aromatase, sulfatase and sulfotransferase. The disadvantage of long-term SERM and SEEM treatment is a frequent occurrence of serious side effects. In the case of SERM, it is vaginal bleeding, endometrial carcinoma, the need for hysterectomy, ischemic cerebrovascular events and venous thromboembolism (Demissie et al. J. Clin. Oncol. 2001, 19, 322). After SEEM application, increased bone breakage, constipation/diarrhea, nausea and vomiting, sleep disturbances, fatigue/weakness, flushing and sweating, vaginal haemorrhage, hair loss, weight changes, depression and others are observed (Eastell et al. J. Clin. Oncol. 2008, 26, 1051). A substantial portion of breast tissue tumors show increased expression of 17βHSD1. It is believed that with modulation of 17βHSD1 activity it would be possible to influence the local E2 level in the affected tissue and thereby regulate the growth of tumor tissue. None of the 17βHSD1 inhibitor has undergone clinical trials yet.
Treatment of prostate cancer is based on a decrease in androgen levels. This can be achieved by surgical or pharmacological (hormonal) castration, or by their appropriate combination. Hormonal therapy consists either of stopping the production of testosterone in testes (LHRH analogues of gonadotrophins), or of blocking the androgen receptor in the prostate cell with antiandrogens (eg. cyproterone acetate). With antiandrogen therapy, side effects are significant, too, including impotence, hot flushes, gynecomastia, mastodynia, digestive problems, depression, fatigue, malaise, and more. A substantial portion of prostate tumors also show increased expression of 17βHSD5. It is believed that with modulation of activity it would be possible to influence the local T level in the affected tissue and thereby regulate the growth of tumor tissue.
A number of 17βHSD inhibitors are described in the literature. Particular emphasis is placed on the preparation of selective, reversible 17βHSD1 inhibitors with minimal or no estrogenic effect. Although research in this area is very intense and involves countless in vitro and in vivo studies, no selective 17βHSD1 inhibitor has so far been in the clinical phase of testing as a potential therapeutic agent for the treatment of estrogen-dependent types of diseases (Poirier, D. Expert Opin. Ther. Patents 2010, 20, 1123).
The 17βHSD1 and 17βHSD5 inhibitors can be structurally divided into two large groups, namely non-steroidal inhibitors and steroid-based inhibitors. Since the present invention discloses inhibitors that are estrone derivatives, only the group of steroid inhibitors of 17βHSD1 and 17βHSD5 will be further discussed. The topic of 17βHSD inhibitors was covered in several reviews (Penning, T. M.; Ricigliano, J. W. J. Enzyme. Inhib. 1991, 5, 165; Poirier, D. Curr. Med. Chem. 2003, 10, 453; Broz̆ic̆ et al. Curr. Med. Chem. 2008, 15, 137; Poirier, D. Anti-cancer Agents Med. Chem. 2009, 9, 642; Day et al. Minerva Endocrinol. 2010, 35, 87).
The following overview will focus on the development in the field of inhibitors of 17βHSD1, especially from 1990 until present. Inhibitory activity against 17βHSD1 has been tested for progestin derivatives, e.g., nomegestrol acetate, medrogestone, tibolone and their metabolites, using the cancerous lines MCF-7 and T-47D (estrogen-dependent breast cancers) at the physiological level of E1. The inhibitors were not selective for 17βHSD1 (e.g., Chetrite et al. J. Steroid Biochem. Molec. Biol. 1996, 58, 525; Chetrite, G. S.; Pasqualini, J. R. J. Steroid Biochem. Molec. Biol. 2001, 76, 95; Shields-Botella et al. J. Steroid Biochem. Mol. Biol. 2005, 93, 1). The effect of Dydrogesterone (Duphaston®) and its 20-dihydro metabolite on the E1 conversion to E2 was also tested (Chetrite, G. S. et al. Anticancer Res. 2004, 24, 1433).
A series of 17 estratrienes fluorinated at the C-17 position was prepared by Deluca et al. and tested for inhibitory activity against the five isoforms of 17βHSD (1, 2, 4, 5, 7). The compounds showed an average inhibitory activity against 17βHSD1 and a poor selectivity against the other isoforms tested. The study of estrogenic potential of substances was not the subject of this study (Deluca et al. Mol. Cell. Endocrinol. 2006, 248, 218).
A series of variously substituted E2-based compounds with inhibitory activity against 17βHSD1 (also tested for isoforms 2 and 3) were prepared in the D. Poirier group. The inhibitor carrying butyl(methyl)thiaheptanamide substituent on the C-6 carbon exhibited 40% inhibitory activity at a concentration of μmol·l−1 (Poirier et al. J. Steroid Biochem. Mol. Biol. 1998, 64, 83). Subsequently, so-called dual inhibitors, simultaneously carrying two pharmacophores in the C-16 position, were also prepared. The best of the prepared derivatives also exhibited anti-estrogenic effects (Pelletier et al. Steroids 1994, 59, 536; Tremblay, M. R.; Poirier, D. J. Chem. Soc., Perkin Trans. 11996, 2765).
Hybrid inhibitors with a steroid skeleton were also prepared, having a side chain of different lengths carrying adenosine on the C-16 carbon. The best compound, EM-1745, is an excellent competitive, reversible inhibitor (Qiu et al. FASEB J. 2002, 16, 1829; Poirier et al. Synt. Commun. 2003, 33, 3183). In 2005, new series of selective hybrid inhibitors of 17βHSD1 were described (also tested for 17βHSD2 isoenzyme). These were E1 (or 2-ethyl-E1) derivatives bearing a —CH2CONHR group at C16 position. The best of the prepared compounds showed the concentration of the substance necessary for 50% inhibition, i.e., IC50, in the range of 27-37 nmol·l−1 at a concentration of E1=2 nmol·l−1 (Lawrence et al. J. Med. Chem. 2005, 48, 2759). Simultaneously, inhibitors with C-16α-bromoalkyl and C-16β-bromoalkyl groups have been developed. Although the compounds were potent inhibitors of 17βHSD1, they were estrogenic. Estrogenicity was later eliminated by modification of the steroid skeleton at C-7 position, but this modification led to a decrease in inhibitory activity (Tremblay, M. R.; Poirier, D. J. Steroid Biochem. Mol. Biol. 1998, 66, 179; Blomquist et al. Endocrinol. 1997, 153, 453; Tremblay et al. Steroids 2001, 66, 821). Other modifications of the C-16 side chain of the steroid skeleton have resulted in a large number of enone, enol, phenol, sulfamate and saturated alcohols. However, improvement in inhibitory activity against 17βHSD1 was not achieved (Ciobanu, L. C.; Poirier, D. Chem. Med. Chem. 2006, 1, 1249).
E1/E2 derivatisation at positions C-7, C-16, C-17 was also studied. E1 derivatives were prepared with a pyrazole or isoxazole ring comprising a C—C bond between C-16 and 17 carbons (Sweet et al. Biochem. Biophys. Res. Comm. 1991, 180, 1057); series of E1/E2 derivatives, bearing a substituted pyrazole ring, comprising a C—C bond between C-16 and 17 carbons, were also tested. IC50 values of derivatives of E1-C16-methylcarboxamides ranged from tens of nmol·l−1 (Allan et al. J. Med. Chem. 2006, 49, 1325). For the group of N- and C-substituted 1,3,5(10)-estratriene-[17,16-c]-pyrazole derivatives, the inhibitory activity was worse, ranging in hundreds of nmol·l−1. The N-substitution of the pyrazole ring, however, suppresses the estrogenicity of these derivatives. The later prepared derivatives had much better IC50 values determined on T-47D cells (WO2004085457; Vicker et al. Chem. Med. Chem. 2006, 1, 464). In summary, E1/E2 derivatisation at positions C-7, C-16, C-17, and the biological activity of the most promising inhibitors is discussed in (Purohit et al. Mol. Cell. Endocrinol. 2006, 248, 199).
E1/E2 derivatization at C-3, C-16, C-17 was also tested. The best of the 17βHSD1 inhibitors tested was 16β-m-carbamoylbenzyl-E2 (E2B), able to reduce proliferation induced by the physiological level of E1 in T-47D ER+ cells by 62%. Cell growth was not stopped by 100% because the substance itself exhibited weak estrogenicity (Laplante et al. Bioorg. Med. Chem. 2008, 16, 1849). Therefore, its 16β,17β-γ-lactone was prepared. The substance was not estrogenic, but its inhibitory activity against 17βHSD1 significantly decreased. The E2B derivative, which carried the bromoethyl chain instead of the 3-OH group, demonstrated that for the successful inhibition of 17βHSD1, the presence of the 3-OH group on the A-ring of the steroid skeleton was not necessary. This substance is not estrogenic, it is a competitive irreversible selective inhibitor, and was tested in vivo on mouse xenograft model with T-47D cells. It was shown that at a dose of 250 μg/day/mouse, the tumor was reduced by 74% after 32 days (WO2012129673, Ayan et al. Mol. Cancer. Ther. 2012, 11, 2096; Maltais et al. J. Med. Chem. 2014, 57, 204).
Messinger et al. prepared a large series of C-15α/β-E1 derivatives, some of which had a hydroxyl group in the C-3 position, and others had its methyl-ether, and showed excellent inhibitory activity. Selectivity and estrogenicity were not discussed (Messinger et al. Mol. Cell. Endocrinol. 2009, 301, 216; WO2005047303, US20050192263). The same authors also patented 17-difluoroestratiens substituted at the C-15 position by a chain bearing in most cases an amide functional group (WO2006125800, US20060281710). Again, this is a large group of substances with high inhibitory activity, usually sufficiently selective for 17βHSD1 (WO2008065100). The authors described a method of measuring estrogenicity, however estrogenicity is not quantified in the document. A series of estratrienes substituted at the C-15 position by triazole derivatives is described in WO2008034796 and US20080146531. It is a very large group of substances with excellent inhibitory activity of about 90% at a concentration of 1 μmol·l−1. Selected derivatives are selective inhibitors of 17βHSD1 (also tested for 17βHSD2 and 3 isoenzymes).
Estratrienes substituted at the C-15 position with triazole derivatives, with a steroid skeleton modified at C-2, 3, 4, 15 and 17 positions, are selective inhibitors of 17βHSD1 (WO2014207311, WO2014207309, WO2014207310).
17βHSD1 inhibitors also include C-2-D-homo-E1 derivatives; the most active is 2-phenethyl-D-homo-E1 with IC50=15 nmol·l−1 (Möller et al. Bioorg. Med. Chem. Lett. 2009, 19, 6740; WO2006003012).
The new 2-substituted estra-1,3,5(10)-trien-17-ones are described in U.S. Pat. No. 7,419,972 (WO2006003013). Their inhibitory activity towards 17βHSD1 is characterized by IC50 values ranging from tens to hundreds of nmol·l−1.
As far as we know to date, C-15 estrone derivatives as 17βHSD1 inhibitors are the subject of only a few of the above-mentioned patents of Solvay Pharmaceuticals (J. Messinger) and Forendo Pharma LTD (L. Hirvela) and one publication (Messinger et al. Mol. Cell. Endocrinol. 2009, 301, 216-224).
In all of these cases, they are structurally very similar substances, but they differ considerably from our derivatives. Our presented derivatives exhibit a more advantageous and complex set of biological properties.
17βHSD5 Inhibitors
C-3,17 and 18-oxirane steroid derivatives have been prepared, but their inhibitory activity and selectivity have not yet been published (Penning et al. Molec. Cell. Endocr. 2001, 171, 137). Selective inhibition of 17βHSD5 has been described for J2404 derivative (Deluca et al. Mol. Cell. Endocrinol. 2006, 248, 218). From the spirolactone series tested, the EM1404 derivative (3-carboxamido-1,3,5-(10)-estratrien-17(R)-spiro-2-(5,5-dimethyl-6-oxo)tetra-hydro-pyran) was the best competitor and selective inhibitor with IC50=3,2 nmol·l−1 and Ki=6,9 nmol·l−1 (Qiu et al J. Biol. Chem. 2007, 282, 8368; WO9946279).
A similar spirolactone prepared by Bydal et al., 3-deoxyestradiol with C-17-dimethyl-spiro-δ-lactone showed IC50=2.9 nmol·l−1. The substance is only negligibly estrogenic and is not androgenic, but selectivity to individual isoenzymes is not discussed (Bydal et al. Eur. J. Med. Chem. 2009, 44, 632). A synthesis of two series of C-17-spirolactone derivatives of androstane was also published. Substances do not bind to ER or exhibit androgenic activity, they inhibit 17βHSD5 in the range of 54-73% at a concentration of 0.3 μmol·l−1 (Djigoué et al. Molecules 2013, 18, 914). Bothe and co-authors have recently introduced a series of estra-1,3,5(10),16-tetraene-3-carboxamide derivatives (WO2013045407, WO2014128108). The compounds carry a variously substituted pyridine ring at the C-17 position and have been introduced as inhibitors of 17βHSD5, IC50<50 nmol·l−1. The synthesis and use of C-3 substituted estra-1,3,5(10),16-tetraenes with a similar IC50 value is presented in WO2014009274.